Turning a Natural Biopolymer-Binding Protein into a Plastic Surface-Recognizing Protein

Abstract

Type A carbohydrate-binding modules (CBMs) preferentially recognize crystalline polysaccharides such as cellulose and chitin, yet some can also bind synthetic plastics, suggesting that their recognition properties can be redesigned through protein engineering. Here, we used directed evolution to alter the substrate specificity of the archaeal chitin-binding protein PfChBD2 toward plastics. Two rounds of evolution, targeting surface residues and conserved aromatic residues, were screened by phage display, yielding mutants with markedly reduced chitin affinity and distinct PET-binding profiles characterized by apparent binding parameters. Structural modeling indicated that substitutions altering surface electrostatics and hydrophobicity contributed to the shift in substrate preference. When fused to a fluorescent tag, the engineered proteins bound several plastics, including PET, PS, PE, and PP, while showing minimal interaction with natural polysaccharides. The proteins also stained microplastics collected from seawater, demonstrating their potential for environmental detection. This study further demonstrates CBMs as evolutionarily adaptable scaffolds capable of recognizing synthetic polymers and highlights the potential of engineered CBM-based probes for microplastic detection, polymer analysis, and biotechnological and environmental applications.